The synergistic effect of EPA and DHA with cyclooxygenase-1 inhibitors on platelet aggregation

J Atherosclerosis Prev Treat. 2023 May-Aug;14(2):57-63| doi:10.53590/japt.02.1047

RESEARCH ARTICLE

Ourania Argyropoulou, Aikaterini N. Tsouka*, Despoina Pantazi*, Ioannis K. Koutsaliaris, Alexandros D. Tselepis

Atherothrombosis Research Centre, Department of Chemistry / Laboratory of Biochemistry, University of Ioannina, Ioannina, Greece

*Equal contribution

 

Abstract

Aim: Consumption of omega-3 fatty acids docosahexaenoic acid (DHA, 22:6ω-3) and eicosapentaenoic acid (EPA, 20:5ω-3) from a variety of foods and supplements could reduce cardiovascular events. The antiplatelet drugs aspirin (ASA) and triflusal, bind to platelet cyclooxygenase-1, thus preventing the biosynthesis of thromboxane A2 from arachidonic acid. ASA, triflusal, and their combinations with DHA or EPA were evaluated against in vitro platelet aggregation induced by the agonists AA, ADP, or TRAP-6.

Materials-Methods: Platelet Rich Plasma (PRP), isolated from blood of healthy volunteers was pre-incubated with acetylsalicylic acid, triflusal, EPA, DHA, or the drug-omega-3 combinations at various concentrations for 10min at 37ºC, prior to activation by AA, TRAP-6, and ADP (0.3mM, 10μM and 6μM, respectively). Platelet aggregation was determined by Light Transmittance Aggregometry.

Results: DHA significantly improves ASA and triflusal’s inhibitory effect towards AA-induced platelet aggregation, while EPA enhanced only the antiplatelet activity of ASA. Moreover, only the combination of ASA with EPA exhibited enhanced inhibitory effect against platelet aggregation induced by ADP. Furthermore, the inhibition of ASA and triflusal on TRAP-6-induced platelet aggregation was enhanced when ASA or triflusal were combined with EPA.

Conclusions: Both DHA and EPA inhibit AA, ADP and TRAP-6-induced platelet aggregation in a dose dependent manner. The results of this study suggest a potentially beneficial use of EPA and DHA supplementation, in conjunction with the antiplatelet drugs ASA and triflusal.

Key words: Aggregation, acetylsalicylic acid, aspirin, eicosapentaenoic acid, docosahexaenoic acid, triflusal

Corresponding author: Professor Alexandros D. Tselepis, Atherothrombosis Research Centre/Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, GR-45110, Tel.: +30 26510 08365, Fax:+30 26510 08677, E-mail: atselep@uoi.gr

Submission: 09.12.2021, Acceptance: 24.04.2023

INTRODUCTION

Polyunsaturated fatty acids (PUFAs) play a key role in reducing the risk of cardiovascular diseases (CVDs)1. PUFAs exhibit cardioprotective2,3 and anti-inflammatory effects and have positive effects in neurological disorders4,5. More specifically, eicosapentaenoic acid (EPA, 20:5-3) and docosahexaenoic acid (DHA, 22:6-3), the most known omega-3 (ω-3) polyunsaturated fatty acids (n-3 PUFAs), are obtained from vegetables and their oils, from eggs, nuts and mainly from fatty fishes and their products, especially salmon, and tuna6,7. There is strong evidence from clinical studies that consumption of these fatty acids in pharmacological doses significantly reduce serum triglyceride levels and may exert a variety of anti-atherothrombotic effects8. Several clinical studies evaluated the effect of DHA and EPA through the years. The most recent studies, are the EVOLVE II (A Prospective Multicenter Trial to Assess the Safety and Effectiveness of the SYNERGY Everolimus-Eluting Platinum Chromium Coronary Stent System (SYNERGY Stent System) for the Treatment of Atherosclerotic Lesion(s)), the ASCEND (A Study of Cardiovascular Events iN Diabetes), the REDUCE-IT (Reduction of Cardiovascular Events with Icosapent Ethyl–Intervention Trial), the EVAPORATE (Effect of Icosapent Ethyl on Progression of Coronary Atherosclerosis in Patients With Elevated Triglycerides on Statin Therapy) and the STRENGTH (Outcomes Study to Assess STatin Residual Risk Reduction With EpaNova in HiGh CV Risk PatienTs With Hypertriglyceridemia) Clinical Trials. More specifically, the EVOLVE II study showed that 2g of PUFAs daily, reduce triglyceride and non-HDL cholesterol levels in patients with severe hypertriglyceridemia (SHTG) and benefit acute pancreatitis and CVD9. Patients with diabetes mellitus but without CVD showed no significant difference in serious vascular effects after ω-3 fatty acids administration, according to the ASCEND Study10. Moreover, the REDUCE-IT study showed that in patients with increased triglyceride levels receiving statins, the risk of CVD death was decreased in the group of patients treated with 2g of EPA twice daily as compared with the placebo group11. Furthermore, the EVAPORATE Study showed that the combination of EPA with statins slowed coronary plaque progression and regression over 18 months12. Finally, the STRENGTH Clinical trial showed that among patients at high CV risk, the addition of ω-3 fatty acids to usual background therapies did not reduce the composite endpoint of major adverse cardiac events13.

Aspirin or acetylsalicylic acid (ASA), a COX-1 inhibitor, remains the primary therapeutic option for CVD prevention, including acute events14–16. ASA acts by blocking the production of thromboxane A2 (TXA2)17,18. Administration of ASA can lead to gastrointestinal toxicity19,20. Another antiplatelet drug very similar to ASA is triflusal. Triflusal inhibits irreversibly the COX-1 activity but to a lesser extent compared with ASA21–23. Due to these characteristics, triflusal exerts a comparable efficacy but with a safer profile compared to ASA24,25. Several clinical trials have designed to highlight triflusal as a promising alternative drug versus ASA14–16,26.

The aim of our study was to investigate the possible synergistic effect of EPA or DHA on ASA- and triflusal-induce inhibition of platelet aggregation induced by arachidonic acid (ΑΑ), adenosine diphosphate (ADP) and thrombin receptor TRAP-6 in vitro. To examine the possible synergistic effect, fatty acids and antiplatelet drugs were used in concentrations that induce a minimum inhibition of platelet aggregation.

MATERIALS AND METHODS

Materials and Reagents

Docosahexaenoic acid (DHA) was purchased from Biosynth Carbosynth, Carbosynth Ltd United Kingdom and Biosynth AG Switzerland) and Eicosapentaenoic acid (EPA) was kindly provide by Libytec Pharmaceutical S.A. The antiplatelet drugs Acetylsalicylate lysine and Triflusal were obtained from Egicalm, Galinos Company, Greece and Galenica SA, Greece, respectively. Arachidonic Acid (A.A.) and Adenosine Diphosphate (ADP) were purchased from Sigma-Aldrich, St. Louis, MO, USA, and the Thrombin Receptor Activating Peptide-6 (TRAP-6) was purchased from Bachem, Bubendorf, Switzerland.

Platelet aggregation studies

The antiplatelet activity of the fatty acids and antiplatelet drugs was studied in platelet-rich plasma (PRP) by the Light transmittance aggregometry (LTA) assay, as we previously described27–29. Briefly, PRP was prepared from citrated blood samples withdrawn from peripheral venous blood of apparently healthy volunteers and the platelet number was adjusted to 250 × 106/mL with homologous platelet-poor plasma (PPP). The platelet aggregatory response to A.A. (300 μM), ADP (6 μΜ) or TRAP-6 (10 μΜ) was studied in aliquots of 0.5 ml PRP at 37 °C under continuous stirring at 1,200 rpm, in a Chronolog Lumi-Aggregometer (model 700 4-channel) equipped with the AggroLink software package. In some experiments, the PRP was pre-incubated for 10 min at 37°C with DHA or EPA, dissolved in Dimethyl sulfoxide (DMSO) or the antiplatelet drugs ASA or triflusal dissolved in sterile water or ethanol (EtOH), respectively, or their combination, before the initiation of the aggregation. In these experiments, the final concentration of EtOH and DMSO was 0.4 % (v/v). The inhibitory efficacy of PUFAs was expressed as IC50 values, in μΜ (the concentration that induces 50% inhibition of maximum platelet aggregation). In control experiments, PRP was incubated in the aggregometer with DHA or EPA for 5 min at 37 °C under continuous stirring to evaluate whether these PUFAs are platelet activators themselves. All aggregation assays were conducted within 3h after blood venipuncture.

Statistics

Values were normally distributed as was checked by the Shapiro-Wilk test. For statistical comparisons we used F test and post hoc analysis was performed using the Bonferroni adjustment to correct for multiple comparisons. A p-value of <0.05 was considered significant.

RESULTS

Effect of DHA and EPA on Platelet Aggregation induced by various agonists

The effect of DHA and EPA on platelet aggregation induced by AA, ADP and TRAP-6 is shown in Figure 1 (A, B, C, respectively). Both DHA and EPA significantly inhibited A.A.-induced platelet aggregation in a dose-dependent manner exhibiting IC50 values of 161.8 and 144.6 μΜ, respectively. The minimum significant inhibitory effect of 20.81±7.56% and 22.52±3.46% by DHA and EPA, respectively, was observed at the concentration of 125 μΜ. Platelet aggregation induced by ADP was inhibited by 20 % at the DHA concentration of 125 μΜ, an effect that was not significantly increased even at the concentration of 1000μΜ (28% inhibition). EPA at the concentration of 125 μΜ inhibited ADP-induced platelet aggregation by 20%, exhibiting a similar potency with DHA, however at concentrations of 500μΜ or higher, it inhibited platelet aggregation by 55%. Finally, both DHA and EPA were potent inhibitors of TRAP-6-induced platelet aggregation exhibiting IC50 values of 89.0 and 94.0 μΜ, respectively whereas a 20 % inhibition was observed at 75 μΜ of DHA or EPA (Fig.1).

Figure 1. (A) Dose-dependent effect of EPA and DHA on platelet aggregation induced by AA (300μΜ). (B) Dose-dependent effect of EPA and DHA on platelet aggregation induced by ADP (6μΜ). (C) Dose-dependent effect of EPA and DHA on platelet aggregation induced by TRAP-6 (10μΜ).

Estimation of the aspirin and triflusal concentration that express a minimum antiplatelet effect

To define the synergistic effect of PUFAs with aspirin or triflusal, we estimated the concentration of these antiplatelet drugs that cause an up to 30% inhibition of platelet aggregation induced by the three agonists. ASA, at a concentration of 25 μΜ inhibited by 20% platelet aggregation induced by Α.Α., ADP, or TRAP-6 (Fig.2). Moreover, triflusal at a concentration of 400μΜ exhibited a 20% inhibition of platelet aggregation induced by A.A., whereas a 20% inhibition of platelet aggregation induced by ADP or TRAP-6 was observed at a triflusal concentration of 500μΜ (Fig. 2).

Effect of the combination of antiplatelet drugs with DHA or EPA on human platelet aggregation induced by A.A.

The combination of ASA (25 μΜ) with DHA or EPA inhibited platelet aggregation by more than 93%, p<0.0001 compared to the inhibition induced by ASA, DHA, or EPA alone (Fig. 2). In addition, the combination of triflusal with DHA induced an 87.6±10.78% inhibition of platelet aggregation, while the triflusal and EPA combination a 20.1±3.46% inhibition of platelet aggregation, p<0.0001 compared to triflusal, DHA, or EPA alone (Fig. 2).

Effect of the combination of antiplatelet drugs with DHA or EPA on human platelet aggregation induced by ADP.

The combination of ASA with DHA induced a 48.30±11.63 % inhibition of platelet aggregation induced by ADP, showing that this combination exhibits a cumulative effect against platelet aggregation induced by ADP. In addition, the combination of ASA with ΕPA caused an inhibitory effect of 60.93±13.48% against platelet aggregation induced by ADP, improving the inhibitory effect of ASA. Moreover, DHA and triflusal exhibited a 31.07±8.98% inhibition of platelet aggregation induced by ADP as well as EPA and triflusal inhibited by 33.89±12.47% the platelet aggregation induced by ADP, showing that both fatty acids did not improve the inhibitory effect of triflusal regarding the platelet aggregation induced by ADP (Fig. 2).

FIgure 2. The effect of DHA (125μΜ) and EPA (125μΜ) in combination with Triflusal (400μΜ) or ASA (25μΜ) on platelet aggregation induced by AA, *p<0.001 compared to DHA or ASA, **p<0.001 compared to DHA or triflusal and ***p<0.001 compared to EPA or ASA. The effect of DHA (125μΜ) and EPA (125μΜ) in combination with Triflusal (500μΜ) and ASA (25μΜ) at platelet aggregation induced by ADP, #p<0.01 compared to DHA or ASA and ##p<0.001 compared to EPA or ASA. The effect of DHA (75μΜ) and EPA (75μΜ) in combination with Triflusal (500μΜ) and ASA (25μΜ) at platelet aggregation induced by TRAP-6, $p<0.008 compared to ASA or EPA. Values represent the mean ± SD from at least 3 different experiments.

Effect of the combination of antiplatelet drugs with DHA or EPA on human platelet aggregation induced by TRAP-6

The combination of ASA with DHA induced a 42.74±10.21% inhibition of the platelet aggregation induced by TRAP-6 showing that this combination exhibits a cumulative effect against platelet aggregation induced by TRAP-6. In addition, the combination of ASA with EPA induced a 63.21±19.89% inhibition of platelet aggregation induced by TRAP-6. Furthermore, the combination of triflusal with DHA exhibited a 43.42±7.80% inhibitory effect towards platelet aggregation induced by TRAP-6 showing a cumulative effect, whereas triflusal with EPA exhibited an inhibitory effect of 62.24±15.17% towards platelet aggregation induced by TRAP-6 showing a synergistic effect. (Fig. 2).

DISCUSSION

Both EPA and DHA show antiplatelet activity against A.A., ADP and TRAP-6-induced platelet aggregation, while EPA is the more potent of the two. The results of this study show a potentially beneficial use of antiplatelet drugs ASA and triflusal in conjunction with EPA and DHA supplementation. A number of in vitro and in vivo studies in mouse models support the idea that omega-3 fatty acids can enhance the activity of other platelet inhibitors.30,31

The combinations of EPA and DHA with ASA yield an enhanced inhibitory activity against platelet activation through the A.A. pathway, that indicate a possible synergistic effect. Long chain omega-3 fatty acids antagonize A.A. as the substrate to produce several eicosanoids, such as prostaglandins, thromboxanes, and leukotrienes.32–35 Inhibition of TxA2 synthesis results in TxA3 production, that exhibits weak pro-aggregatory properties.35–37

Furthermore, the combination of EPA and ASA yields an enhanced antiplatelet effect against ADP induced platelet activation. E-resolvins (RvE1), the specialized pro-resolving mediators (SPMs) that occur after the metabolism of EPA, selectively engage with ADP activated platelets, indicating a possible mechanism of antiplatelet activity.38,39 Similarly, the DHA and triflusal combination resulted in an enhanced inhibitory effect toward platelet activation induced by ADP. Furthermore, EPA potentiates the inhibitory effect of ASA and triflusal toward TRAP-6 dependent platelet aggregation.

The underlying mechanisms regarding the pleiotropic activity and the possible beneficial effects of EPA and DHA are currently under investigation by our laboratory team. According to our knowledge, this is the first in vitro study of a possible triple antiplatelet effect of ASA and triflusal with DHA or EPA.

Acknowledgements

The authors thank the Atherothrombosis Research Centre (ARC) of the University of Ioannina for providing access to the laboratory equipment and facilities. The authors gratefully thank Libytec Pharmaceutical S.A. for kindly providing the Eicosapentaenoic acid that was used in the present study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The present work was co-funded by the European Union (ERDF) and Greek national funds through the Operational Program “Competitiveness, Entrepreneurship and Innovation”, under the call “STRENGTHENING RESEARCH AND INNOVATION INFRASTRUCTURES” (project code: 5002803).

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